U.S. patent application number 15/329704 was filed with the patent office on 2017-08-10 for light microscope having a sample stage for cryomicroscopy.
This patent application is currently assigned to Leica Mikrosysteme GmbH. The applicant listed for this patent is European Molecular Biology Laboratory, Leica Mikrosysteme GmbH. Invention is credited to John BRIGGS, Leander GAECHTER, Reinhard LIHL, Martin SCHORB.
Application Number | 20170227752 15/329704 |
Document ID | / |
Family ID | 53546634 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170227752 |
Kind Code |
A1 |
LIHL; Reinhard ; et
al. |
August 10, 2017 |
LIGHT MICROSCOPE HAVING A SAMPLE STAGE FOR CRYOMICROSCOPY
Abstract
In a light microscope (1) for cryomicroscopy, encompassing at
least an objective (2) and a sample stage (3) having a cutout (7)
for a coolable holder (8) for a sample carrier mount, the cutout
(7) being covered by a cover (6), the sample stage (3) is
displaceable in two horizontal directions (4). The cover (6) rests
floatingly on the sample stage (3), and the objective (2) passes
through a cutout (12), corresponding to the objective (2), in the
cover (6). The method for cooling a holder (8) for a sample carrier
mount in a light microscope (1) for cryomicroscopes, by means of a
flow of liquid nitrogen through a cooling conduit (15), open at at
least one end, in the holder (8), is notable for the fact that the
quantity of liquid nitrogen is dimensioned so that all of the
nitrogen is present in gaseous form at at least one open end (16)
of the cooling conduit (15).
Inventors: |
LIHL; Reinhard; (Vienna,
AT) ; BRIGGS; John; (Heidelberg, DE) ; SCHORB;
Martin; (Ladenburg, DE) ; GAECHTER; Leander;
(Oberriet SG, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Leica Mikrosysteme GmbH
European Molecular Biology Laboratory |
Vienna
Heidelberg |
|
AT
DE |
|
|
Assignee: |
Leica Mikrosysteme GmbH
Vienna
AT
European Molecular Biology Laboratory
Heidelberg
DE
|
Family ID: |
53546634 |
Appl. No.: |
15/329704 |
Filed: |
July 15, 2015 |
PCT Filed: |
July 15, 2015 |
PCT NO: |
PCT/EP2015/066105 |
371 Date: |
January 27, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G02B 21/28 20130101;
G02B 21/34 20130101; G02B 21/02 20130101; G02B 21/26 20130101 |
International
Class: |
G02B 21/28 20060101
G02B021/28; G02B 21/34 20060101 G02B021/34; G02B 21/02 20060101
G02B021/02; G02B 21/26 20060101 G02B021/26 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 29, 2014 |
DE |
10 2014 110 723.3 |
Claims
1. A light microscope (1) for cryomicroscopy, comprising an
objective (2) and a sample stage (3) having a cutout (7) for
receiving a coolable holder (8) for holding a sample carrier mount,
the cutout (7) being covered by a cover (6), wherein the sample
stage (3) is displaceable in two horizontal directions (4); the
cover (6) rests floatingly on the sample stage (3); and the
objective (2) passes through a cutout (12) in the cover (6).
2. The light microscope according to claim 1, wherein the cutout
(12) in the cover (6) is a circular hole whose diameter is less
than 2 mm larger than a diameter of a portion of the objective (2)
which passes through the cutout (12) in the cover (6).
3. The light microscope according to claim 1, wherein the sample
stage (3) is coolable.
4. The light microscope according to claim 3, wherein at least one
of the sample stage (3) and the holder (8) has a nitrogen internal
cooling system.
5. The light microscope according to claim 1, wherein the sample
stage (3) has an opening (5) for delivery of a sample carrier mount
to the holder (8).
6. The light microscope according to claim 5, further comprising a
closure device (10) operable to close the opening (5).
7. The light microscope according to claim 1, wherein the cover (6)
comprises a transparent double panel (6').
8. A method for cooling a holder (8) for holding a sample carrier
mount in a light microscope (1) for cryomicroscopy according to
claim 1, comprising: providing a cooling conduit (15) in the holder
(8), the cooling conduit (15) having at least one open end (16),
guiding a flow of liquid nitrogen through the cooling conduit (15)
in the holder (8), wherein the flow of liquid nitrogen is
dimensioned so that all of the nitrogen is present in gaseous form
at the at least one open end (16) of the cooling conduit (15).
9. The method according to claim 8, wherein a metal frit element
(17) is placed at the at least one open end (16) of the cooling
conduit (15).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is the U.S. national phase of
International Application No. PCT/EP2015/066105 filed Jul. 15,
2015, which claims priority of German Application No. 10 2014 110
723.3 filed Jul. 29, 2014, the entirety of which is incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to a light microscope for
cryomicroscopy, encompassing at least an objective and a sample
stage having a cutout for a holder, coolable on the microscope, for
a sample carrier mount, the cutout being covered by a cover.
BACKGROUND OF THE INVENTION
[0003] Cryofixation is a sample preparation method often used in
electron microscopy. In this, a water-containing sample is frozen
very rapidly to a temperature below -150.degree. C. (cryofixed),
i.e. it is cooled very quickly in order to avoid the formation of
ice crystals. Cryofixation has proven to be particularly suitable
for investigations of structural biology. The specimens to be
investigated, for example cells, enzymes, viruses, or lipid layers,
are thereby embedded in a thin, vitrified layer of ice. The great
advantage of cryofixation is that the biological structures can be
maintained in their natural state. For example, a biological
process can be halted at an arbitrary point in time by cryofixation
and investigated in that vitrified state, for example using a
cryo-electron microscope but also in a light microscope with
corresponding sample cooling; cryo-light microscopy serves
principally for ascertaining relevant regions in the sample, those
regions being noted and then viewed in more detail in a
cryo-electron microscope.
[0004] The frozen samples, which as a rule are located on an
electron microscopy sample carrier known per se, for example a grid
or a pin for scanning electron microscopy, must be conveyed, under
the aforesaid cryogenic conditions and with water excluded, into
corresponding sample carrier mounts that can then be conveyed into
appropriate holders in the aforesaid microscopes. A typical sample
carrier mount for use in conjunction with the present invention has
been disclosed, for example, in the document U.S. Pat. No.
8,395,130 B2, in which a grid that serves as a sample carrier and
carries the cryogenic sample can be secured with clip elements in a
corresponding mount. For this, liquid nitrogen is made available,
for example, in a Styrofoam container, in which container the
necessary manipulation steps for conveying the grid into the sample
carrier mounts were accomplished. The formation of cryogenic
nitrogen gas from the liquid nitrogen on the one hand ensures the
necessary low temperature and on the other hand creates an
anhydrous atmosphere, so that contamination of the samples with
water, and consequently with ice crystals, is prevented.
[0005] In order not to impair the quality of the frozen samples, it
is very important that they be transferred in cooled and
contamination-free fashion between the processing devices being
used, for example cryofixation device, freeze fracture apparatus,
or coating apparatus, and the analytical devices, here especially a
cryo-light microscope and cryo-electron microscope.
[0006] One vacuum cryo-transfer system is represented, for example,
by the "Leica EM VCT100" system of the manufacturer Leica
Microsystems, with which a sample carrier mount can be removed from
a liquid nitrogen-cooled manipulation container and conveyed into
the observation chamber, likewise cooled with liquid nitrogen, of
an electron microscope.
[0007] A sample carrier mount could also be transferred with such
an apparatus into the cooled sample stage of a cryo-light
microscope. It is self-evident to one skilled in the art that the
term "coolable" in conjunction with the present invention means
that the components referred to as "coolable" are coolable using
special cooling systems that are provided on the microscope or
attached to it in order to ensure cooling of the cryogenic samples
during observation through the microscope.
[0008] It is apparent that in the context of sample preparation for
cryomicroscopy, considerable effort is expended in order to avoid
contamination of the sample with water and consequently with ice
crystals. Clearly, this must also be ensured after transfer of the
sample or sample carrier or sample carrier mount into the working
region of a cryo-light microscope; sample stages for light
microscopes, which comprise a cutout in which a coolable holder for
a sample carrier mount is located, are available for this purpose
in the existing art. In addition to cooling of the holder, with
these known sample stages the cutout is supplied or flushed with
liquid nitrogen or with cryogenic nitrogen gas, the cutout being
covered with a cover in order to limit nitrogen consumption and
prevent the entry of moisture from the air; in this, a slight
overpressure is established by the nitrogen gas that is delivered
or is produced from the liquid nitrogen, so that ambient air is
forced out of the cutout and prevented from flowing in. With these
known approaches the cover is transparent, since it is located in
the beam path of the light microscope. The objective of the light
microscope can be brought in, however, only until it is directly
against the cover, thus producing a not insignificant spacing
between the objective and the sample, which in turn results in a
small aperture and, associated therewith, low resolution.
SUMMARY OF THE INVENTION
[0009] The object on which the invention is based is therefore that
of overcoming the disadvantages of the existing art in cryo-light
microscopy and enabling a large aperture for viewing while reliably
shielding the sample from moisture.
[0010] In order to achieve this object, a light microscope of the
kind recited previously is refined according to the present
invention in such a way that the sample stage is displaceable in
two horizontal directions; that the cover rests floatingly on the
sample stage; and that the objective passes through a cutout,
corresponding to the objective, in the cover. Thanks to the
floating mounting of the cover, the horizontal displaceability of
the sample stage can be retained even through the objective passes
through the cover in order to bring the objective directly against
the sample so as to achieve a large aperture.
[0011] According to a preferred embodiment of the present
invention, the cutout in the cover is a circular hole whose
diameter is less than 2 mm larger than the diameter of that part of
the objective which is to be brought through the cover. These
dimensions make it possible, after placement of the cover onto the
cutout in the sample stage, to guide the objective through the
cutout in the cover without great difficulty and at the same time
to build up a sufficiently high overpressure with tolerable
nitrogen consumption in order to reliably keep moisture away from
the sample.
[0012] As already mentioned, in cryo-light microscopy a cooled
holder for a sample carrier mount is arranged in a cutout of the
sample stage. To avoid radiation of heat from the region of the
sample stage onto the sample, the light microscope according to the
present invention is preferably refined in such a way that the
sample stage is coolable. The result is that the environment of the
sample is also reliably cooled, and the desired state of the sample
is continuously maintained.
[0013] The sample stage and/or the holder for a sample carrier
mount preferably have a nitrogen internal cooling system. A cooling
system of this kind is based on directing liquid or cryogenic
gaseous nitrogen through a corresponding cooling conduit in the
interior of the relevant parts, thereby resulting in highly
efficient cooling of those parts. The cooling system can be exactly
regulated by way of the flow rate of the nitrogen and an electric
heating system.
[0014] The sample stage preferably has an opening for delivery of a
sample carrier mount to the holder for a sample carrier mount. Such
an opening is preferably provided laterally on the sample stage,
and allows insertion of a sample or a sample carrier mount into the
sample stage.
[0015] In order to prevent moist air from getting to the sample in
the manipulation container even when no insertion is being made
into the sample stage, however, the invention is preferably refined
in such a way that the opening for delivery of a sample carrier
mount is closable by means of a closure device. The closure device
can preferably be configured as a slider, a flap, or the like.
[0016] Visual monitoring of the observation region is essential for
the user even though the cutout in the sample stage is covered. The
invention is therefore preferably refined in such a way that the
cover comprises a transparent double panel. The transparency makes
it possible to see through the cover, while the provision of a
double panel prevents undesired condensation problems due to the
temperature difference between the interior of the sample stage and
the environment.
BRIEF DESCRIPTION OF THE DRAWING VIEWS
[0017] The invention will be explained in more detail below with
reference to an exemplifying embodiment depicted schematically in
the drawings, in which:
[0018] FIG. 1 is an overall view of a cryo-light microscope
according to the present invention;
[0019] FIG. 2 is a perspective depiction of the sample stage of the
cryo-light microscope according to the present invention, without
the cover of the cutout in the sample stage;
[0020] FIG. 3 is a perspective depiction of the sample stage of the
cryo-light microscope according to the present invention, with the
cover of the cutout in the sample stage;
[0021] FIG. 4 is a partial section view of passage of the objective
through the cover; and
[0022] FIG. 5 is a partial section view of the holder for a sample
carrier mount.
DETAILED DESCRIPTION OF THE INVENTION
[0023] In FIG. 1, the number 1 designates a cryo-light microscope
according to the present invention that encompasses, inter alia, an
objective 2 and a sample stage 3. Sample stage 3 can be displaced
not only vertically but also in two horizontal directions in
accordance with arrows 4, in order to ascertain relevant regions of
a sample in sample stage 3. The sample stage possesses an opening 5
for delivery of a sample carrier mount to holder 8 for a sample
carrier mount, which is depicted in FIG. 2. A cover 6 rests
floatingly on sample stage 3 and can therefore be freely displaced
thereon. Objective 2 is guided through the cutout in cover 6 and
carries it along upon displacement of sample stage 3 in the
direction of arrows 4. Cover 6 encompasses a transparent double
panel 6'.
[0024] FIG. 2 shows a cutout 7 in sample stage 3, in which cutout a
holder 8 for a sample carrier mount is located. Holder 8 for a
sample carrier mount possesses clip elements 9 that serve for
clamping immobilization of a sample carrier mount (not depicted).
Holder 8 for a sample carrier mount possesses an infeed 10 and an
outlet 10' for cryogenic nitrogen, so that holder 8 for a sample
carrier mount can be cooled. This is described in further detail in
conjunction with FIG. 5. FIG. 2 furthermore shows opening 5 for
delivery of a sample carrier mount. A closure device 11 for closing
off opening 5 is embodied in the form of a slider 11.
[0025] FIG. 3 now shows that cover 6 completely covers cutout 7 in
sample stage 3 in order to minimize the entry of moisture from the
environment. Cutout 12 in cover 6 allows objective 2, depicted in
FIG. 1, to be guided through the plane of cover 6 and thus to be
brought directly against the sample in holder 8 for a sample
carrier mount. A sleeve 13 is placed on cutout 12 and further
enhances the sealing effect between objective 2 and cover 6.
Because cover 6 rests floatingly on sample stage 3, it can readily
be carried along by objective 2 and thus displaced on sample stage
3.
[0026] FIG. 4 shows the passage of objective 2 through cutout 12.
Sleeve 13 rests more or less against objective 2 and thereby
enhances the sealing effect between objective 2 and cover 6. It is
further evident from FIG. 4 that cover 6 possesses a double panel
6' in order to forestall condensation problems.
[0027] It is apparent from FIG. 5 that a nitrogen conduit 14 is
guided through infeed 10 so that cryogenic liquid nitrogen can be
guided into cooling conduit 15 of holder 8 for a sample carrier
mount. Cooling conduit 15 has an open end 16 onto which a metal
frit element is placed. Nitrogen leaves cutout 7 through outlet
10'. The above-described nitrogen internal cooling system in holder
8 for a sample carrier mount ensures reliable cooling of the
samples, and forces out ambient moisture.
PARTS LIST
[0028] 1 Cryo-light microscope [0029] 2 Objective [0030] 3 Sample
stage [0031] 4 Arrows [0032] 5 Opening [0033] 6 Cover [0034] 6'
Transparent double panel [0035] 7 Cutout [0036] 8 Sample holder
[0037] 9 Clip elements [0038] 10 Infeed [0039] 10' Outlet [0040] 11
Closure device/slider [0041] 12 Cutout [0042] 13 Sleeve [0043] 14
Nitrogen conduit [0044] 15 Cooling conduit [0045] 16 Open end
[0046] 17 Metal frit element
* * * * *